Low-Level Quantitation of N-Nitroso Dabigatran Etexilate Impurity in Dabigatran Etexilate Mesylate API Using the Agilent 6495C LC/TQ

Significance of the topic

Nitrosamines are potent mutagens and potential carcinogens that can form as drug substance–related impurities (NDSRIs) in pharmaceutical APIs. Regulatory agencies require trace-level control of these impurities to ensure patient safety and product quality.

Objectives and study overview

This study aimed to develop and validate a highly specific, sensitive, and reproducible method for quantifying N-nitroso dabigatran etexilate impurity in dabigatran etexilate mesylate API at picogram levels using Agilent 6495C triple quadrupole LC/TQ coupled to an Agilent 1290 Infinity II LC system.

Methodology and instrumentation

• Sample preparation: API dissolved at 5 mg/mL in optimized diluent (MeOH/ACN with 0.05% NH₄OH); working standards from 0.01 to 1 ng/mL; recovery samples spiked at 0.1 ng/mL.
• Chromatography: Agilent 1290 Infinity II LC with Poroshell HPH-C18 (150 × 3 mm, 2.7 µm); mobile phases: 1 mM ammonium trifluoroacetate/formic acid in water (A) and MeOH/ACN (B); gradient 55–15% A over 15 min; flow rate 0.5 mL/min; column at 40 °C.
• Mass spectrometry: Agilent 6495C triple quadrupole with Jet Stream ionization in positive MRM mode; optimized source (gas flows/temperatures, capillary voltage 3.2 kV) and compound parameters; MRM transitions 657.3>364.1, 433.2, 627.3 m/z.
• Diverter valve: built-in valve to divert high-concentration API eluent to waste, preventing MS contamination during early and late gradient phases.
• Data acquisition and analysis: Agilent MassHunter Optimizer, Qualitative and Quantitative Analysis v12.0; calibration using 1/x² weighting.

Main results and discussion

• Limits of detection and quantitation: LOD = 6 pg/mL (0.0012 ppm), LOQ = 10 pg/mL (0.002 ppm) with S/N >70:1 and >190:1 (rms noise).
• Linearity: 0.01–1 ng/mL (0.002–0.2 ppm), R² = 0.9993 with 1/x² weighting.
• Specificity: clear separation of impurity peak from API; no matrix interference observed.
• Accuracy: standard recoveries between 80% and 120% across calibration range.
• Precision: %RSD ≤2.65% for six injections across two preparations; retention time and area reproducibility robust.
• Recovery: 97–110% in three different API lots, demonstrating method robustness for quality control.

Benefits and practical applications

• Enables routine QC screening of low-level N-nitroso dabigatran etexilate impurity in API production.
• Complies with regulatory requirements for nitrosamine limits in pharmaceuticals.
• Diverter valve strategy reduces instrument contamination and maintenance.
• Approach adaptable to other mutagenic impurity quantitation in drug substances.

Future trends and potential uses

• Expansion to comprehensive nitrosamine panels across multiple drug products.
• Integration of high-resolution MS for confirmatory analysis of unknown impurities.
• Automation of sample prep and data workflows to increase throughput and consistency.
• Development of universal stabilizing diluents for acid-sensitive APIs.
• Implementation of real-time impurity monitoring during manufacturing for continuous quality assurance.

Conclusion

The developed Agilent 6495C LC/TQ MRM method delivers sub-picogram sensitivity, excellent linearity, specificity, precision, and recovery for quantifying N-nitroso dabigatran etexilate in API. Its robustness and compliance with regulatory limits make it ideal for routine pharmaceutical quality control.

References

1. FDA guidance on NDSRI acceptable intake limits, CDER, 2023
2. Bernardi R. et al., Stability-indicating LC method for dabigatran etexilate, J. AOAC Int., 2013
3. FDA guidance: LC-HRMS method for nitrosamine impurities in ARB drugs
4. FDA guidance: LC-HRMS method for nitrosamine impurities in metformin, 2023
5. Schlingemann J. et al., The landscape of potential nitrosamines in pharmaceuticals, J. Pharm. Sci., 2023
6. Karun Mutha V.V.S.R.N. & Katari B.V.N.R., Hydrolytic degradation of dabigatran etexilate mesylate, Anal. Chem. Lett., 2018